Amacrine cells are interneurons in the vertebrate retina that help to shape the visual signals that ganglion cells send to other visual centers in the brain. The ultimate goal of the research proposed here is to understand how the signaling molecule nitric oxide functions in the retina to shape amacrine cell signaling and to contribute to normal visual processing in the retina. This proposal specifically addresses the role of nitric oxide in controlling chloride levels in amacrine cells. This is highly relevant to neuronal function because the level of chloride in a cell determines the effect of traditionally inhibitory neurotransmitters such as GABA and glycine on cells at synapses. If chloride is kept low in a neuron, then GABA and glycine will mediate inhibitory signals but if chloride levels are increased, excitatory signals can be generated by these neurotransmitters. The research proposed here is based on our previous work demonstrating that nitric oxide causes release of chloride from internal stores and has the power to temporarily switch inhibitory synapses into excitatory synapses. Although this effect of nitric oxide has been clearly demonstrated, very little is understood about the underlying mechanisms and how this action affects retinal function. To learn more about this, the following three specific aims are proposed: Specific Aim 1: To elucidate the mechanisms underlying the nitric oxide-dependent redistribution of chloride in amacrine cells, Specific Aim 2: To discover the transporter(s) involved in regulating the distribution of internal chloride, Specific Aim 3: To determine the effects of endogenously generated NO. Elucidation of the mechanisms underlying the action of nitric oxide on amacrine cell chloride (Aims 1 and 2) will be achieved using cultures containing identified GABAergic amacrine cells derived from the chick retina. Recordings of amacrine cells in retinal slices will be used to explore the impact of nitric oxide on amacrine cell function. These studies will involve a combination of electrophysiological recordings, digital imaging using ion- sensitive fluorescent dyes and siRNA gene knockdown methods. What is learned here about the role of nitric oxide in regulating internal chloride fluxes may be a valuable component of understanding pathologic conditions related to defects in cellular cytosolic chloride management such as Dent's disease, and osteopetrosis. Understanding how NO regulates amacrine cell function will contribute to our understanding of retinal signal processing. PUBLIC HEALTH RELEVANCE: The proposed research will expand our understanding of the regulation of cellular chloride levels and the actions of the signaling molecule nitric oxide. These factors are relevant to the normal and disease states of cells in multiple tissue types including kidney, bone and brain. In particular we will discover how nitric oxide regulates cellular chloride levels and how this regulation contributes to plasticity in the retina and other parts of the brain. [unreadable] [unreadable] [unreadable]